Monitoring the effect of subunit assembly on the structural flexibility of human alpha apohemoglobin by steady-state fluorescence

A single energy transfer distance, between the sole intrinsic tryptophanyl donor [14 (A12)] and a nonfluorescent sulfhydryl acceptor probe (4-phenylazophenylmaleimide, PAPM) attached to the only cysteine [104 (G11)], has been employed to examine the effect of subunit assembly on the structure of the heme-free human-hemoglobin. Efficiencies of energy transfer were measured in 0.05 M potassium phosphate buffer,pH 7.0, at 5°C, and the structural flexibility of-apohemoglobin, in the absence and presence of human-heme-containing chains, was examined by a steady-state solute quenching technique. The quenched efficiencies (E^O) and Förster distances (R₀^O) were analyzed by least-squares to determine the goodness of fit (_R²) for the assumed distribution parameters: average distance ¯r and half-widthhw. Data for-apohemoglobin in the absence and presence of^h chains yielded values for ¯r of 18 and 22 Å andhw of 20 and 8.5 Å, respectively. Although the increase in ¯r for-apohemoglobin in the presence of^h chains was presumably a consequence of additional quenching from the heme moiety, the change in the half-width strongly indicated a decrease in the flexibility of the-apohemoglobin chain within the assembled protein. A transition in structural flexibility similar to that demonstrated here may be an important aspect of human hemoglobin assembly.